It has previously been demonstrated that rats trained on the peak-interval procedure to associate two different cues with two different fixed interval schedules will generate a scalar peak function at an intermediate time when presented with the compound cue. This response pattern has been interpreted as resulting from the simultaneous retrieval of different temporal memories, and a consequential averaging process to resolve the ambiguity. In the present set of studies, we investigated the role that serotonin 1a receptors play in this process. In Experiment 1, rats were trained on a peak-interval procedure to associate the interoceptive states induced by saline and the 5-HT1a agonist, 8-OH-DPAT, with a 5 s or 20 s fixed-interval schedule signaled by the same tone cue . While peak functions following administration of saline were centered at the appropriate time (5 s or 20 s), peak functions following administration of the agonist were centered around 7 s, irrespective of the reinforced time during training, suggesting agonist-induced disruption in selective temporal memory retrieval, resulting in increased ambiguity regarding the appropriate time at which to respond. In Experiment 2, rats were trained in a peak-interval procedure to associate a tone cue with a 10 s fixed interval and a light cue with a 20 s fixed interval. Administration of the 5-HT1a antagonist, WAY-100635, had no impact on timing when single cues were presented, but altered the intermediate, scalar, response to the stimulus compound, suggesting antagonist-induced disruption in the processes used to deal with temporal memory ambiguity. Together, these data suggest that manipulations of 5HT transmission at the 5-HT1a receptor cause changes in the temporal pattern of responding that are consistent with alterations in temporal memory processes and responses to temporal ambiguity. Adapting behavior to the temporal regularities in the environment is critical for the efficient utilization of resources. This capacity requires organisms to perceive the duration between events and outcomes, store these durations in memory, and temporally control their behavior using these memories when re-exposed to the predictive events. The ability to perceive and behave with respect to time in the seconds to minutes range, interval timing, has frequently been studied using reproduction procedures, such as the peak-interval procedure . In thisThe vast majority of peak-interval timing studies have utilized test trials in which the duration to be timed is explicitly indicated to the subject. In other words, there is a “correct” response time and location to maximize the likelihood of reinforcement. Considerably less work has examined how subjects deal with ambiguity regarding the appropriate response time. Such ambiguity can occur when animals are trained to time multiple durations. For example, in the “bi-peak” or “tri-peak” procedure, a single cue is used to indicate the availability of reward following one of two or three randomly selected durations , and whiWe, and others, have conducted a number of studies investigating how animals respond to ambiguous temporal information resulting from the presentation of compound stimuli. Under certain circumstances, subjects will behave as though they have computed a weighted average of the component durations, and then timed this average in an otherwise normal manner . For exaA number of subsequent studies have confirmed and extended these basic findings. We have also demonstrated that rats can be trained in the peak-interval procedure to use interoceptive cues to predict the time of reinforcement. Using a drug-discrimination approach, in which the administration, and subsequent interoceptive effects, of a pharmacological agent serve as the discriminative stimulus for operant responding , Kurti eA classic finding in the interval timing literature is that dopaminergic psychostimulants, such as amphetamine, methamphetamine, and cocaine can increase the speed of an internal clock , 2006. FAlthough ad libitum access to food and water until the experiment began. One week prior to training, food access was restricted and the rats were maintained for the remainder of the experiment at approximately 85–90% of their free-feed weight, adjusted for growth. Prior to beginning training, approximately ten 45 mg grain pellets  were given to each rat in their home cage to acclimate them to the reinforcer used in the operant chambers. All animals were kept on a 12-h light:dark cycle (lights on at 8:00 AM – rats were trained and tested during the light cycle). Food rations were given shortly after each daily session. Unless noted otherwise, all training and testing sessions lasted 120 min. All procedures were approved by the Institutional Animal Care and Use Committee of Villanova University.Twenty male Sprague-Dawley rats (Harlan), approximately 90 days of age at the onset of the experiment, were subjects. The animals were housed two per cage and given Training and testing took place in standard operant conditioning chambers described in detail previously . Three n8-OH-DPAT was dissolved in 0.9% physiological saline (250 μg/ml), and either the drug or vehicle  was administered subcutaneously just prior to each training and test session at a volume of 1.0 ml/kg. Drug and vehicle injections were counterbalanced across days in 2-day blocks, with the order in each block randomized.Rats were given 12 sessions of fixed-interval training in which the first nosepoke response made either 5 s (short) or 20 s (long) following onset of a 1 kHz auditory cue was reinforced with a 45 mg grain pellet, at which point the cue terminated, ending the trial. Responses made prior to the completion of the fixed interval had no programmed consequence. The FI duration in place for each session depended on whether the rat was given saline or the 5-HT1a agonist immediately beforehand. In group Saline-short/Drug-long, saline was always paired with the short 5 s FI and 8-OH-DPAT was always paired with the long 20 s FI. In group Drug-short/Saline-long, 8-OH-DPAT was always paired with the short 5 s FI and saline was always paired with a 20 s FI. Rats in group Saline-short/Drug-long were trained and tested during the spring, and rats in group Drug-short/Saline-long were trained and tested during the summer. Trials were separated by a uniform 60–80 s inter-trial interval.Following these 12 FI sessions, rats were given 16 sessions of peak-interval training, with the same drug-duration association. The first five trials of each session were non-reinforced probe trials, and the remaining trials were composed of 75% FI trials, and 25% probe trials, resulting in means of 16.7 (1.5) and 16.2 (1.4) probe trials per session on saline and drug days, respectively. Although data from FI trials were not saved, the expected mean number of FI trials per session would be 34.3. Probe trials lasted 60–80 s and terminated independently of responding. We ran the initial non-reinforced probe trials to allow assessment of temporal expectation associated with the saline/drug injection, prior to any feedback about the correct duration for that day.Following these PI sessions, the rats were given three test sessions in which all trials were non-reinforced probes, and no feedback was provided, resulting in a mean of 51.4 (0.5) probe trials per session. Prior to each of these test sessions, the rats were given an injection of saline, the training dose of the 5-HT1a agonist (250 μg/kg), or an intermediate dose of the 5-HT1a agonist (125 μg/kg), with the order of administration counter-balanced across rats. In between each of these test sessions, a saline PI training session and a drug PI training session were conducted.0 + A ∗ (1/(1 + exp(-1∗((x - B + C/2)/D)))) ∗ (1-(1/(1 + exp(-1∗((x - B - C/2)/E))))), as used previously  for the saline peaks, and 0.934 (0.0856) for the drug peaks. The CV was computed by dividing peak spread by peak time.Nosepoke entries were binned into 1-s bins to create peak-response functions. Data from the last eight PI training sessions (four drug and four vehicle) were pooled for analysis. Data from the test sessions  were analyzed separately. Due to the skewed pattern of responding frequently seen following drug administration (see Results), rather than use a standard Gaussian to describe the data, we fit  the pooled responses with a dual asymmetric sigmoid function, Y = Yeviously . Y0 is tSingle trial analyses were conducted by fitting the response pattern on individual trials with a series of three flat lines to capture the initial low response rate, a high response rate, and a terminal low response rate . Start apost hoc tests were conducted when relevant. The current session’s drug state (Drug) served as the within subject factor.Because the two groups were trained and tested in different months (spring and summer), repeated measure analysis of variance (ANOVA) were conducted for each group separately using SPSS (v24), with an alpha level set at 0.05. LSD Figure 1, separately for the two groups and split by administered drug; group Saline-short/Drug-long’s peak functions are shown Figure 1A and group Drug-short/Saline-long’s peak functions are shown in Figure 1B. Figure 1C depicts these same data as a proportion of maximal response rate over time to allow easier comparison of the timing of responses irrespective of changes in response rate, and to allow comparison across groups.The response rates on drug days in the Saline-short/Drug-long group during the PI training sessions were extremely low, preventing an assessment of temporal control prior to the rats receiving feedback. Therefore, the data from probe trials following these initial five trials were analyzed. Response rates as a function of time on these PI sessions are plotted in Figures 1A,C – solid red line) peaked around the short peak time of 5 s, whereas rats in group Drug-short/Saline-long peaked around the long peak time of 20 s . In contrast, following administration of 8-OH-DPAT, rats appeared to peak later than 5 s in the Drug-short/Saline-long group , and peak earlier than 20 s in the Saline-short/Drug-long group . Specifically, the mean peak time following saline administration in group Saline-short/Drug-long was 4.6 s (0.9) and the mean peak time following saline administration in group Drug-short/Saline-long was 16.8 s (2.5). The mean peak time following 8-OH-DPAT administration in group Saline-short/Drug-long was 7.6 (3.5) and the mean peak time following 8-OH-DPAT in group Drug-short/Saline-long was 5.8 s (1.4).As can be seen, following administration of saline, rats peaked at a time that corresponded to their condition; rats in group Saline-short/Drug-long  = 5.69, p< 0.05; Drug-short/Saline-long: F = 168.47, p< 0.001].Repeated measure ANOVAs conducted on peak times with Drug as a within-subject factor demonstrated significant effects for both groups , but not in the Drug-short/Saline-long group [F < 1]. These differences in CV in the Saline-short/Drug-long group are likely due to changes in response times over the session due to relearning, as they were not seen during the test sessions (see below).The CV  in group Saline-short/Drug-long was 1.95 (0.61) following saline and 4.08 (2.42) following 8-OH-DPAT. In group Drug-short/Saline-long, the CV was 1.81 (0.34) following 8-OH-DPAT, and 1.74 (0.41) following saline. A repeated measures ANOVA on relative spreads revealed significant effects of Drug in the Saline-short/Drug-long group .We also assessed whether poke duration  changed as a result of drug administration. Mean poke durations for each subject were 0.49 s (0.15) and 0.51 s (0.10) for the short and long associated durations, respectively, in the Saline-short/Drug-long group. For the Drug-short/Saline-long group, poke durations were 0.29 s (0.06) and 0.32 s (0.09) for the short and long associated interoceptive cues, respectively. Repeated measure ANOVAs revealed no differences in either group . Further, one sample t-tests demonstrated that the peak times on saline were not different from the programmed criterion duration of 5 s [Saline-short: t(9) = 1.86] and 20 s [Saline-long: t(9) = 0.39]. In contrast, the peak times on drug were different from their respective programmed criterion durations .These apparent differences in peak times were confirmed with a repeated measures ANOVA, with Drug as a within-subject factor. There was a main effect of Drug in both groups [Saline-short/Drug-long: F = 1.11]. We note that because the two groups were tested in different months, this between group comparison should be interpreted cautiously.This phenomenon, that the rats could discern the two drugs states and retrieve the appropriate corresponding duration when the drug signaled the short duration, but would not be able to respond at the appropriate duration when the drug signaled the long duration, coupled with the remarkable similarity in the peak functions following 8-OH-DPAT administration across the groups, is similar to some of the partial or incomplete averaging phenomena seen before . TherefoF’s < 1.3].The CVs were 1.82 (0.86) for the saline data in the Saline-short/Drug-long group, 2.46 (1.59) for the drug data in the Saline-short/Drug-long group, 1.82 (0.67) for the drug data in the Drug-short/Saline-long group, and 1.82 (0.54) for the saline data in the Drug-short/Saline-long group. Repeated measure ANOVAs with Drug as a within-subject factor yielded no significant results .Rats in the Saline-short/Drug-long group poked for 0.50 s (0.13) following saline administration, and for 0.54 s (0.13) following 8-OH-DPAT administration. Rats in the Drug-short/Saline-long group poked for 0.35 s (0.09) following saline administration and 0.33 s (0.08) following 8-OH-DPAT administration. A repeated measures ANOVA with Drug as the within-subject factor, revealed no significant effects . Post hoc comparisons indicated significant differences between saline and the intermediate dose (ps < 0.005) in both groups, but not between the intermediate and full training dose in either group (ps > 0.25). The coefficients of variation were 1.75 (1.09) for the Saline-short/Drug-long group, and 2.33 (0.87) for the Drug-short/Saline-long group. No significant differences in relative spreads were found: .All rats were also given an intermediate dose of the drug (125 μg/kg) during a non-reinforced probe session in order to assess how rats would respond to an interoceptive state that was presumably similar to both the training drug state and the training saline state. However, given that the full dose of the training drug did not generate peak response functions that were in the appropriate location, interpretation of these sessions is not immediately clear. For completeness, the peak times following the intermediate dose were 7.3 s (2.1) in the Saline-short/Drug-long group, and 8.3 (3.0) in the Drug-short/Saline-long group. Repeated measure ANOVAs with Drug  as a factor indicated main effects in both groups: . The mean start and stop times in each condition are presented in F = 151.98, p< 0.001] as well as an effect of Drug , but no interaction . For the Drug-short/Saline-long group, there was the expected main effect of Transition , a main effect of Drug , as well as a Transition × Drug interaction . Post hoc pairwise comparisons indicated significant differences in both start times and stop times across drug states (both p< 0.001).As with the peak functions, the start and stop times were most similar to the reinforced durations following saline, and were at intermediate locations following administration of 8-OH-DPAT. A repeated measures ANOVA on the Saline-short/Drug-long data with Transition  and Drug as within subject factors revealed the expected main effect for Transition ethyl]-N-2-pyridinyl-cyclohexan ecarboxamide (WAY-100635), a selective 5-HT1a antagonist, or saline, and tested on their response to the presentation of the non-reinforced compound stimulus . Based on the findings in Experiment 1, suggesting increased temporal memory ambiguity and averaging behavior following 8-OH-DPAT administration, we expected that blocking the 5-HT1a receptor would lead to an increase in temporal memory selectivity on the ambiguous compound trials, thereby disrupting the scalar temporal averaging that is consistently seen under these conditions.Once training was completed, rats were given Ten male Sprague-Dawley rats (Harlan) approximately 90 days of age at the onset of the experiment were subjects. All aspects of housing and the conditioning chambers were identical to that described in Experiment 1.WAY-100635 was dissolved in 0.9% physiological saline and administered intraperitoneally immediately prior to half of the test sessions as a fixed proportion of the animals’ body weight (1.0 mg/kg). Drug and vehicle  injections were counterbalanced across days. Injections began only after all animals completed the training phase of the experiment.Rats were trained on a peak-interval procedure to time two different durations (10 s and 20 s) associated with two different discriminative signals . Counter-balancing of the modality-duration relationship was not done for the reasons described above. After stable, temporally controlled responding was obtained to the single cues, the rats were tested on both the trained stimuli and novel compound stimuli (simultaneous tone + light) while under the influence of the drug (WAY-100635) and vehicle. Initial training sessions took place overnight during 12-h sessions. Following acquisition of the criterion durations, training was continued using 2-h daily sessions. Rats were run 7 days a week at approximately the same time each day. Initial overnight training sessions took place during the rats’ dark cycle, while the 2-h training and testing sessions occurred during the rats’ light cycles. All rats were given injections of the vehicle prior to training during the final two training sessions to minimize non-specific effects during testing. The composition of training and testing sessions is listed below.Each trial began with onset of either a 10 s “short” stimulus  or a 20 s “long” stimulus (illumination of the house light). Following the target duration, the first nosepoke in the center aperture terminated the stimulus and a 45 mg grain pellet was delivered to the food magazine. There was no programmed consequence for responding early, and the signals stayed on until a reinforced response occurred. The probability of a tone or light trial was equal (50% each) during fixed-interval training, and trial type for this and all subsequent training/testing sessions was selected at random with replacement. Trials were separated by a uniform variable inter-trial interval of 40–60 s.Peak-interval training was identical to fixed interval training, except for the addition of non-reinforced probe trials, which terminated independently of responding following a variable interval that was 3–4 times the “long” criterion duration . Reinforcement probability was set such that the reinforcement rate for the two stimuli was equivalent, which is necessary for temporal averaging to occur . To thisTesting was identical to peak-interval training, except for the addition of non-reinforced probe trials in which the simultaneous compound stimuli (tone + light) was presented, and rats were given an injection of drug or vehicle immediately prior to each session. Due to the short terminal half-life for WAY-100635 , 0.872 (0.067), and 0.707 (0.196), for the short, compound and long peaks, respectively, on saline sessions and mean R2 values of 0.800 (0.194), 0.755 (0.166), and 0.460 (0.300) for the short, compound, and long peaks, respectively, on drug sessions. The CV was computed as peak spread/peak time. Single trial analyses were conducted as described in Section “Experiment 1.” Summary data are presented as mean (SD). Repeated measures ANOVAs were conducted with Duration  and Drug  as factors. Post hoc comparisons were performed as simple effects  using the overall error term from the omnibus ANOVA. Greenhouse-Geisser correction was utilized if violations of sphericity occurred.Data from the first 30 min of each testing session were pooled for analysis. Given the low numbers of trials and the low response rate following WAY-100635 administration, which hindered assessment of temporal control, responding was binned in 2-s bins. In contrast to Experiment 1, because peak responding was generally symmetrical, pooled response rate functions were fit with a 4-parameter normal distribution, to obtain measures of peak rate (amplitude), peak time (mean), and peak spread (standard deviation). This 4-parameter function did an acceptable job fitting the data, with mean As a result of a computer malfunction, the rats did not complete the second test session and all data from the session was lost. Rats were consequently given two additional training sessions prior to the resumption of testing. In total, there were a mean of 28.9 (5.4) short probe trials, 9.9 (3.6) long probe trials, and 16.6 (4.8) compound probe trials per rat.Figure 5A. To allow a better visualization of the temporal pattern of responding independent of the change in response rate, the same data are replotted after being normalized by maximal response rates in Figure 5C.Mean response rate as a function of time  from the saline sessions are plotted in As can be seen, the peak functions for the two component cues were centered near the programmed fixed intervals of 10 s and 20 s, and the long peak function was broader than the short peak function, as to be expected given the scalar property. Specifically, the mean peak time for the short tone cue was 11.5 s (2.2) and it was 20.1 s (4.1) for the long light cue. The mean peak spread was 7.3 s (1.1) for the short cue and 10.5 s (4.7) for the long cue. As a result, relative spread (CV) was 0.65 (0.10) for the short cue and 0.52 (0.19) for the long cue. Additionally, the mean amplitude was 1.9 (1.2) responses per second for the short peak function, and 1.7 (0.9) responses per second for the long peak function.Presentation of the stimulus compound (tone + light) resulted in a normal shaped peak at a location midway between the two programmed fixed intervals, with a mean peak time of 15.8 s (2.5), and a mean peak spread of 9.1 (1.8), resulting in a CV of 0.58 (0.07). The average amplitude of the compound peak was 2.5 (1.0) responses per second.Figure 5B, and the same data after being normalized by maximal response rate are replotted in Figure 5D. As can be seen, the administration of the serotonergic 1A antagonist appeared to result in a decrease in response rate , but no change in the temporal control associated with the component cues. The mean peak time for the short tone cue was 12.0 s (2.9) and 20.3 s (5.0) for the long light cue. The mean peak spread was 8.4 s (4.0) for the short cue and 8.5 s (3.9) for the long cue. As a result, the CV (peak spread/peak time) was 0.78 (0.35) for the short cue and 0.44 (0.22) for the long cue.Mean response rate as a function of time  from sessions following administration of WAY-100635 are plotted in Figure 5D). The mean peak time of the compound response function fell at 17.8 s (4.6), with a peak spread of 9.2 s (1.9), resulting in a CV of 0.53 (0.10). Inspection of individual subject’s response functions suggested different response patterns to the compound cue in different rats, with compound responding overlapping either the short or long response function. Indeed, the standard deviation (across subjects) of the compound peak time following drug administration was almost twice that of the compound peak time following saline administration.Presentation of the compound cue again resulted in a peak function that appeared to be of greater amplitude than the component cues. However, in contrast to the compound peak following saline administration, the peak function following WAY-100635 administration appeared to fall closer to the long, light function, nearly overlapping it  and drug . As can be seen, the compound peak functions fell in between the component peaks following saline administration, but overlap one of the component peaks following WAY-100635 administration. Inspection of the distribution of peak times for the compound cue reveals that administration of the drug led to a flat or perhaps bimodal distribution, whereas the compound peak time distribution following saline administration was clearly unimodal . In contrast, the shape of the distributions of peak times for the single tone and light cues did not appear to be impacted by drug administration .Support for a difference in responding to the compound cue can be seen in p> 0.10). Therefore, we conducted a repeated measures ANOVA with Duration and Drug as within-subject factors. Results demonstrated a main effect of Duration , but no effect of Drug [F = 2.22] and no interaction (F< 1). Post hoc comparisons across Duration were all significant . Similarly, a repeated measures ANOVA on CVs revealed only an effect of Duration , with the CV for the short cue being larger than the CV for the compound and long cue (ps < 0.05), but no difference between the compound and long CVs (p> 0.10). There was no effect of Drug (F< 1) and no interaction (F< 1). This failure of the scalar property has been seen in our compounding studies before  = 34.83, p< 0.001], as peak rates declined following administration of WAY-100635, and a significant effect of Duration , but no interaction (F< 1). The effect of Duration was due to the compound peak rate being larger than both the short (p< 0.001) and the long (p< 0.05) peak rates, whereas these component peak rates did not differ (p> 0.10).The change in the breadth of the compound peak time distribution did not impact the normality of the data as indicated by a non-significant test of normality  = 450.0, p< 0.001], the expected main effect of Duration , and a main effect of Drug , as the transition times were later following drug. The Transition × Duration interaction was also significant , with the long start times being different than the short and compound start times, and all stop times being different from one another, except for the compound and long. No other interactions were significant , Drug × Duration: F< 1, Transition × Drug × Duration: F = 1.80].As expected, the single trial transition times progressed in an orderly fashion, largely bracketing the corresponding peak times. A repeated measures ANOVA with Transition , Drug , and Duration  as factors was conducted. There was the expected a main effect of Transition  as stop times were relatively less variable than start times, as seen previously  = 9.36, p< 0.005], as the relative variability was largest with the short (tone) cue, and smallest with the long (light) cue. There was no main effect of Drug [F < 1]. There was also a significant Transition × Duration interaction , resulting from the long start time CV being less variable than the short or compound start time CVs. No other interactions were significant . Critically, there was no evidence that the compound CV was larger than the component CVs, suggesting that rats are not switching back and forth between timing the short cue and timing the long cue on different compound cue trials.A repeated measures ANOVA with Transition, Duration, and Drug as factors demonstrated a main effect of Transition  versus [(long-compound)/(long-short)]. If a rat’s compound peak time falls exactly in between its short and long peak time, this similarity index would equal 0.5, whereas if it falls at the same time as one of the component peak times, the similarity index would equal 0. Negative values result if the compound peak time is shorter than the short peak time or longer than the long peak time. Following saline administration, the median compound similarity index was 0.21 (interquartile range = 0.20), whereas following administration of WAY-100635, the median similarity index was 0.02 (IQR = 0.79). These statistics are shown in Figure 7E. We note that the error bars  are quite large following drug administration, and result from the fact that several of the rats’ compound peak functions fell outside the range of their component peak functions, providing further evidence of a 5-HT1a induced disruption of temporal memory processes on compound cue trials. A paired t-test demonstrated that the relative distance between the compound peak and the closest component peak was smaller following WAY-100635 than saline . In contrast, there was no difference in the absolute distance between the short and long peak times following drug administration [t(9) = 0.14]. As the scalar property of temporal perception  = 2.45, p< 0.05], whereas there was no change in the absolute distance between the short and long peak times [t(9) = 0.27].These data suggest that blocking 5-HT1a receptors lowers the response vigor, but does not impact the temporal control of behavior in response to the trained component cues. However, the overlap between the compound and component cues, and the increase in the breadth of the distribution of compound peak times suggests WAY-100635 may be altering the processes by which rats retrieve multiple temporal memories when presented with the simultaneous component cues. Alternatively, the retrieval processes may operate normally, but the strategies used to deal with the memory discrepancies may have changed. The rats could be switching from an integration-like process to a selection-like process , and/or rception , as wellrception , indicatFigure 7 shows the average peak functions for rats with short-like and long-like compound peaks following saline administration  and following drug administration .Rats whose compound peak time was closer to the short peak time (using the above similarity index) were classified as having short-like compounds, whereas those whose compound peak time was closer to the long peak time were classified as having long-like compounds. As can be seen, there was more similarity between the rats’ compound functions and the short or long component functions following drug than following saline. Under saline, five rats had compound peak times more similar to the short peak, and the other five had compound peak times more similar to the long peak. Under WAY-100635, four rats had compound peak times more similar to the short peak, and six rats had compound peak times more similar to the long peak time. Intriguingly, the component peak that the compound cue was most similar to was not consistent between saline and WAY-100635 sessions, as seven of 10 rats switched which peak the compound cue was more heavily weighted toward. Specifically, four of the five rats with short-like compounds following saline had long-like compounds following drug administration, and three of the five rats with long-like compounds following saline had short-like compounds following WAY-100635.The results from the current experiment suggest that blocking serotonergic transmission at the 5-HT1a receptor does not change temporally controlled behavior in response to single cues (aside from lowering response rates), but it does alter the pattern of responding when multiple cues are presented as a simultaneous compound. Specifically, when presented with a compound cue, rats will typically respond at an intermediate duration with a normally shaped, scalar response form . Indeed,What mechanism might be responsible for these results? One possibility is that blocking the 5-HT1a receptor may have altered the relative salience of the two component cues. Alternatively, the effect of WAY-100635 may be to alter the averaging or integration process, perhaps by changing the relative weight given to each cue. Previous work from our lab has demonstrated that rats can flexibly weight or bias one temporal memory over the other. This change in the weight given to one cue over the other occurs when one of the cues has greater relative value , when thAlthough the mechanisms by which this alteration in temporal control occurs cannot be conclusively identified by our data, some evidence supports a change in the relative weighting of the component cues, rather than a change in perceptual salience. First, the peak amplitude was greater in response to the compound cue than it was to either of the component cues following both saline and WAY-100635 administration. If blocking the 5-HT1a receptor resulted in the compound stimulus being perceived as just one of the component cues due to salience differences, this should result in only one of the corresponding temporal memories being chosen, and there should have been no change in peak rate. Secondly, unpublished pilot data from our lab using drug-naïve subjects, in which we examined the response to the compound cue after manipulations to the volume of the auditory cue, suggest that there were no changes in temporal averaging behavior as a result of presumed changes in auditory salience.Across two experiments, we found that manipulations of 5HT transmission at the 5-HT1a receptor caused changes in the temporal pattern of responding. When jointly considered, results from these experiments are consistent with alterations in temporal memory retrieval and the response strategy used in the face of temporal ambiguity. In Experiment 1, the administration of a 5-HT1a agonist resulted in peak functions centered at a time between the fixed intervals that the rats were trained on, and which were positively (rightward) skewed as though the rats were influenced by both temporal memories. In other words, the rats’ responses following 8-OH-DPAT suggested increased ambiguity about the expected time of reinforcement. In contrast, in Experiment 2, administration of a 5-HT1a antagonist resulted in a disruption of temporal averaging behavior in response to the compound cue, as though the rats’ temporal expectations became less ambiguous.While temporal averaging behavior, as inferred from the production of a scalar peak function at an intermediate duration, is often seen under conditions of ambiguity such as stimulus compounding, this response pattern has been found to depend on the component cues having equivalent value . In contIn contrast to the equivalent reinforcement probabilities, and consequential difference in value for the two durations in Experiment 1, in Experiment 2, the cue for the long duration (light) signaled a higher reinforcement probability than the cue for the short duration (tone). Specifically, the probability of the short cue being reinforced after 10 s was 25%, whereas the probability of the long cue being reinforced after 20 s was 50%, with the remaining trials being non-reinforced probes. As a result, the reinforcement rates (at least up to the time of expected reward) was equal (25%/10 s = 50%/20 s). Under these conditions, presentation of the simultaneous compound stimulus (tone+light) results in a unimodal, normally shaped, scalar peak function centered at an intermediate duration, although the precise location of the peak is typically biased toward the longer duration . This biSerotonin functioning has been broadly implicated in various aspects of cognition . HoweverThese selective effects are consistent with past investigations of serotonin’s role in interval timing. In a review, Intriguingly, serotonin has been recently implicated in playing a modulatory role in multisensory integration . AdminisTo summarize, the studies we described found that administration of serotonergic drugs modulated the use of temporal memories under conditions involving ambiguity. Specifically, use of the 5-HT1a agonist 8-OH-DPAT as a discriminatory cue produced responses influenced by durations learned following saline administration. In contrast, the 5-HT1a antagonist WAY-100635 prevented the complete integration of memories associated with two different cues when these were presented as a compound. Together these results suggest activity of the 5-HT1a receptor plays an important role in retrieval of temporal memories and the resolution of conflicting information.ZS helped to design and conduct the experiments, analyze the data, and write the manuscript. SC helped to design and conduct the experiments and analyze the data. LH helped to conduct the experiments and analyze the data. MM helped to design the experiments, analyze the data, and write the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We found that DPK-060 formulated in acetate buffer or poloxamer gel caused a marked reduction of bacterial counts of Staphylococcus aureus in vitro . We also found that DPK-060 in poloxamer gel significantly suppressed microbial survival in an ex vivo wound infection model using pig skin and in an in vivo mouse model of surgical site infection . Encapsulation of DPK-060 in different types of lipid nanocapsules or cubosomes did not improve the bactericidal potential of the peptide under the applied test conditions. No reduction in cell viability was observed in response to administration of DPK-060 in any of the formulations tested. In conclusion, the present study confirms that DPK-060 has the potential to be an effective and safe drug candidate for the topical treatment of microbial infections; however, adsorption of the peptide to nanocarriers failed to show any additional benefits.Antimicrobial peptides, also known as host defense peptides, have recently emerged as a promising new category of therapeutic agents for the treatment of infectious diseases. This study evaluated the preclinical  The rapidly increasing resistance toward conventional antibiotics has accelerated research efforts to identify new and non-conventional anti-infective therapies , which was developed for the treatment of skin infections. DPK-060 is a chemically synthesized peptide structurally derived from human protein kininogen, where three tryptophan residues have been added to the C-terminal end of the endogenous 17-amino acid sequence  was produced using Fmoc solid phase technology at Bachem AG, Bubendorf, Switzerland. The peptide was identified by electrospray ionization mass spectrometry and the purity was assessed by HPLC. The purity of the peptide used in all studies was 98.5%. Bactroban, 2% ointment  was used as control antibiotics in in situ gelling poloxamer formulation  and methyl paraben  were dissolved in 20 mM acetate buffer pH 5.5 in an ice bath for 1–2 h until the polymer was completely dissolved (23 wt% poloxamer 407 and 0.03 wt% methyl paraben). For formulations containing DPK-060 without a nanocarrier, DPK-060 was first dissolved in acetate buffer and then added to the polymer solution while stirring. The final concentrations of poloxamer 407 and methyl paraben were 17 wt% and 0.02 wt%, respectively; the final concentrations of DPK-060 were 0.25 wt%, 0.5 wt%, and 1.0 wt%, corresponding to approximately 1.0 mM, 2.0 mM, and 4.0 mM, respectively.® WL1349; kindly provided by Gattefossé S.A., Saint-Priest, France), and macrogol 15 hydroxystearate . Briefly, lecithin (75 mg), triglycerides (846 mg), macrogol 15 hydroxystearate , sodium chloride (90 mg), and 20 mM acetate buffer pH 5.5 (3 ml) were mixed and homogenized under magnetic stirring. The resulting emulsion was then heated to 90°C and cooled to 60°C 2 times, and then heated to 90°C and cooled to 78°C, to obtain reversible emulsion phase inversions. After the temperature of 78°C (the phase inversion temperature) had been reached, an irreversible shock was induced by a rapid dilution with 12.5 ml 4°C purified water to reach room temperature. For monolaurin (ML)-LNCs, lecithin was replaced by monolaurin ; monolaurin (300 mg), triglycerides (930 mg), macrogol 15 hydroxystearate (770 mg), sodium chloride (90 mg), and 20 mM acetate buffer pH 5.5 (3 ml) were mixed. The resulting emulsion was then heated to 60°C and cooled to 20°C 2 times, and then heated to 60°C and cooled to 37°C. After the temperature of 37°C (the phase inversion temperature) had been reached, the dispersion was rapidly diluted with 20°C purified water to a volume of 10 ml. The particle size of the LNCs and ML-LNCs was in the range of 50–80 nm and 30–45 nm, respectively, with a polydispersity index < 0.1.Lipid nanocapsules (LNCs) were prepared as described previously  DPK-060 in acetate buffer at a ratio of 3:1 LNCs/ML-LNCs:DPK-060 solution, for 2–3 h [sufficient for peptide molecules to achieve a dynamic equilibrium between the LNCs and the surrounding medium  in an ice bath for 60 min, which resulted in a homogenous viscous liquid. The final concentration of poloxamer 407 was 17 wt%; the final concentrations of DPK-060 were 0.25 wt%, 0.5 wt%, and 1.0 wt%, corresponding to approximately 1.0 mM, 2.0 mM, and 4.0 mM, respectively. The final concentrations of LNCs/ML-LNCs were five-fold higher than the DPK-060-concentrations, i.e., 1.25 wt%, 2.5 wt%, and 5.0 wt%.The adsorption efficiencies of DPK-060 in LNCs and ML-LNCs were 33.8 ± 3.5% and 33 ± 5%–42 ± 2%, respectively,  with cubic phase structure, i.e., cubosomes, were prepared as described previously  using a minimum microbicidal concentration (MMC) assay as described  was performed as previously described  with 0.25 ml of each test item and 1.48 ml of BHIdil.Time-kill assay against Release of DPK-060 from the different formulations was monitored through dialysis as described previously  were mounted in Franz diffusion cells  with a 0.64 mmThe effect of DPK-060 in different formulations on cell viability was determined in compliance with OECD Test Guideline 439 by using the EpiDerm Skin Irritation Test  according to manufacturer's instruction.S. aureus (ATCC 29213) was evaluated in an ex vivo wound infection model using pig skin as previously described , and sample collection/analysis for surgical site infection model were carried out in female mice of CD1 strain as previously described .The injury, infection with escribed ., 2014. The treatment-related systemic toxicity was assessed by observing general behavior and clinical signs including body posture, central excitation, mood, and motor activity. The local tolerability was assessed by observing incidence of erythema and oedema.ex vivo and in vivo studies refer to the total concentrations of DPK-060 in the formulation and do not take into consideration the potential differences in the release pattern.The doses of DPK-060 used in post-hoc test, with a value of P <0.05 considered statistically significant. The statistical analyses were performed using SPSS Statistics .Statistical significance between the groups was calculated using one-way ANOVA followed by LSD The MMC for DPK-060 in acetate buffer or when formulated with different nanocarriers in poloxamer gel was in the range of 1–5 μg/ml . No antiDPK-060 in acetate buffer or in poloxamer gel displayed bactericidal effect in time-kill assay at concentrations equal or higher than 2 μg/ml with a significant reduction in the CFU numbers detected after 3 h of incubation . DPK-060The release of the peptide from DPK-060-loaded LNCs and cubosomes in poloxamer gel was first investigated by dialysis method. The results reveal that after 24 h of incubation, 100% of DPK-060 was released from the LNCs in poloxamer gel as well as from the poloxamer gel without any nanocarriers . In contThe peptide release efficiency from DPK-060-loaded LNCs in poloxamer gel was further investigated by Franz diffusion cell studies. The results reveal identical release profile for DPK-060 in poloxamer gel with or without encapsulation of the peptide into LNCs: approximately 40% of DPK-060 was released within 3 h of incubation and full release (100%) was observed at 10 h .ex vivo pig skin model. The formulations were administered 2 h post-infection with S. aureus, and the bacteria were harvested 4 h post-treatment  was investigated in an reatment . DPK-060reatment . The DPKreatment .ex vivo antimicrobial effect of selected formulations  was further studied in the pig skin model after a longer follow-up period, i.e., the bacteria were harvested 24 h after the treatment  was also studied in a murine reatment . The samreatment . DPK-060reatment . Notablyreatment .in vitro EpiDerm Skin Irritation Test, the test item is considered to display an irritating potential in case the viability is reduced by >50% of the values measured for negative control sample. All the formulations tested, with or without DPK-060, displayed the cell viability of ≥90% of the control sample.In connection to the local application of the formulations in the surgical site infection model in mice, with or without DPK-060, no systemic toxicity or local tolerability concerns were visually observed in any of the mice. According to in vitro bactericidal action against S. aureus with activities in the micromolar range. In vitro, different AMPs including DPK-060, when adsorbed to nanoparticles, have shown enhanced antimicrobial activity both against planktonic bacteria and in reducing biofilm growth  and Gram-positive bacteria were significantly lower in eczematous lesions of patients with atopic dermatitis after 14 days of treatment with DPK-060 1% ointment .In addition to in vitro studies when different AMPs were encapsulated into LNCs or cubosomes  and the reason for low efficacy of DPK-060-loaded LNCs in vivo remains elusive.Protection against proteolytic degradation by infection-affiliated enzymes has earlier been reported in in vitro EpiDerm Skin Irritation Test. Furthermore, we found no visible signs of systemic toxicity or local irritation in murine surgical site infection model in connection to the administration of DPK-060-containing formulations. This is consistent with the results of the clinical trial, where no serious adverse events (SAE) were reported after dermal application of DPK-060 in atopic dermatitis patients, whereas adverse events (AEs) were observed at similar frequency in the DPK-060 and placebo groups .We did not observe any significant reduction in cell viability with DPK-060 in any nanocarrier formulation tested when assessed by ex vivo and in vivo assessments, which is considered a limitation of the experimental design. Repeated application over several days, and a longer follow-up period, would closer resemble the clinically relevant situation and may lead to different results in terms of both efficacy and safety.In conclusion, the present study confirms that DPK-060 has the potential to be an effective and safe drug candidate for the topical treatment of microbial infections; however, under the applied test conditions, the adsorption of the peptide to nanocarriers failed to show any additional benefits. Notably, in this study, a single administration of the test formulations, combined with a short follow-up time, was used both in The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.in vivo and in vitro studies, scientific input, performed the in vivo experiments. LR: planning of studies, scientific input, responsible for the formulation studies. AU, JJ, TA, RR, and CB: performed in vitro studies and scientific input. LB and PT: performed formulation studies and scientific input. PKS: planning of in vitro and in vivo studies, scientific input. PS: planning of in vitro studies and scientific input. MM: overall responsible for the studies and corresponding author.JH: experimental planning and was responsible for At the time of the investigation, MM was employed at Promore Pharma AB and she continues to work for the company at consultancy basis. Promore Pharma is a biopharmaceutical company that develops peptide-based product candidates aimed for the bioactive wound care market. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pre-eclampsia (PE) is the most common pregnancy complication affecting 2–8% of all pregnancies. PE could lead to maternal and prenatal morbidity. Imbalanced cytokine network and altered levels of several inflammatory and anti-inflammatory cytokines have been reported in PE. Because of scare information regarding the roles of IL-17 and IL-35 in PE, the current study aimed to investigate the serum level of these cytokines in a group of Iranian women suffering from PE.Serum samples were collected from 100 pre-eclamptic and 100 healthy pregnant women. Patients and controls were matched for age, ethnicity and body mass index. The level of IL-35 and IL-17 were evaluated by ELISA technique. T test and one-way ANOVA with Tukey Post-Hoc test were used for analysis and p<0.05 were assumed significant.The serum level of IL-35 was increased in pre-eclamptic subjects as compared with healthy pregnant women (p<0.001). There was no significant difference in the serum level of IL-17 between pre-eclamptic and healthy pregnant women (p=0.73). Moreover, the results of the present study also showed that the pregnant women with severe pre-eclampsia had higher level of IL-35 in their sera when compared to those with mild form of the disease (p<0.001). In addition, the serum level of IL-35 was significantly elevated in women with higher proteinuria (p<0.001).Based on the our results, it seems that elevated levels of IL-35 in sera of pre-eclamptic women might work as a marker to evaluate the severity of the preeclampsia. Pre-eclampsia (PE) is the most common pregnancy complication affecting about 2–8% of pregnancies. PE is the main cause of pregnancy related death in developed countries. In addition to morbidity and mortality in pregnant women, PE could lead to prenatal death, preterm birth and also intrauterine growth restriction . ClinicaIL-35 as a member of IL-12 family was first introduced as an anti-inflammatory cytokine mainly produced by Tregs . Recentlgr protein in 24-hour urine along with at least 140 mmHg systolic or 90 mmHg diastolic blood pressure. Moreover, based on the blood pressure and proteinuria, two groups of cases were selected. 54 women with at least 5 gr proteinuria and blood pressure above 160/110 mmHg were selected as severe PE group and 46 were labeled as mild PE group. Women with history of blood pressure before pregnancy, autoimmune disease, malignancy, and active infection were excluded from the study. Inclusion criteria for control group were the same as cases except for PE symptoms. It should be noted that all the patients had received medications. This study was approved by the local Ethics Committee of Shiraz University of Medical Sciences, Iran (IR.SUMS. REC.1395.S513). After signing the informed consents, 2 ml of peripheral blood were collected from all participants. Sera were separated and stored in aliquots at −70°C till performing the ELISA tests.This study was conducted on 100 women diagnosed with PE as cases and 100 healthy pregnant women as the control group. Cases were selected among women who referred to Hafez and Zynabiyeh gynecology hospitals affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. Inclusion criteria for cases were the presence of at least 1+ dipstick or 0.3 μl of serum samples were added to intended wells and incubated overnight at room temperature. After that plate was washed and then biotin-antibody was added and incubated for an hour at 37oC. Upon a triplicate washing, HRP-avidin was added to wells and incubated for another hour at 37oC. After that, wells were washed 5 times and substrate-TMB was added to the wells and incubated in dark at 37oC for 15 min. At final step, stop solution was added to wells and the optical density of each well was checked at 450 nm using ELISA reader. The sensitivity of the IL-17A kit was 1 pg/ml. In case of IL-35, human IL-35 ELISA kit  was used according to manufacturer’s instructions. The whole process was similar to IL-17A evaluation with one exception where after adding the serum samples, plates were incubated for 2 hr at 37oC. The sensitivity of IL-35 kit was 15.6 pg/ml.In order to evaluate the serum level of IL-17 and IL-35, enzyme-linked immunosorbent assay (ELISA) method was utilized. IL-17 was assessed using Human IL-17A (homodimer) ELISA Ready-SET-Go  according to manufacturer’s instructions and recommended concentrations. Briefly, 100 SPSS statistical software, version 16  and oneway ANOVA with proper post hoc tests and student T test were used for data analysis and P-values less than 0.05 were considered statistically significant. Moreover, graphs were designed using ghraphPad PRISM software version 5 .vs. 29.11±5.56). Moreover, there were no significant differences between severe and mild preeclamptic women regarding age (29.65±6.1 vs 30.36±5.6). Based on the level of blood pressure and proteinuria, 54 and 46 cases were diagnosed as severe and mild preeclampsia, respectively. The level of blood pressure was significantly different between cases and controls as well as between severe and mild group . MoreovThe mean serum levels of IL-17A in pregnant women with preeclampsia and healthy pregnant women were 2.79±1.26 and 2.86±1.69, respectively. There was no significant difference in the level of IL-17A among women with mild (3±1.2) or severe preeclampsia (2.5±1.29) when compared with control group . The reIn the next step, the relationship between the levels of IL-35 and IL-17A with blood pressure and proteinuria was compared in patients. Results indicated that the mean level of IL-35 was significantly higher in patients with 4+ proteinuria as compared to those with less than 4+ proteinuria (p<0.001) . There wpg/ml while the level of this cytokine is reported in a range of 6.65–17 pg/ml by Ozkan and Coa et al.  and a Th17 related inflammatory cytokine (IL-17) were evaluated in a group of Iranian women with pre-eclampsia. The results of the present study indicated that the level of IL-35 was significantly increased within sera samples from pre-eclamptic women and also the increase was related to the severity of the disease. In addition, it was observed that pre-eclamptic women with higher level of proteinuria had elevated level of IL-35. It is well known that PE is a disease of placentation. Indeed, it is believed that a poor placentation in the beginning of pregnancy will lead to PE after the 20th week of gestation . To suppa et al. . Regardia et al. . In caseic women . In lineic women . Interesic women . Moreoveic women . The latic women . The conic women –33. Thesic women . As mentic women . If the  and a ThIn all, although high level of IL-35 may be associated with pre-eclampsia and severity of the disease but due to lots of unanswered problems, it is not suitable for evaluating the onset or severity of PE. Also, in case of its role in pathology of PE, there are lots of questions remained to be answered.


Campylobacter fetus fetus and C. fetus venerealis subspecies allow the differential diagnosis of bovine genital campylobacteriosis. The hydrogen sulfide production, for example, is a trait exclusive to C. fetus fetus and C. fetus venerealis biovar intermedius. This gas that can be biochemically tested can be produced from L-cysteine (L-Cys). Herein, we report a novel multiplex-PCR to differentiate C. fetus based on the evaluation of a deletion of an ATP-binding cassette-type L-Cys transporter that could be involved in hydrogen sulfide production, as previously described. A wet lab approach combined with an in silico whole genome data analysis showed complete agreement between this L-Cys transporter-PCR and the hydrogen sulfide production biochemical test. This multiplex-PCR may complement the tests currently employed for the differential diagnosis of C. fetus.Phenotypic differences between  Campylobacter fetus is best known as a major veterinary pathogen that has a detrimental effect on reproductive efficiency of herds. However, in humans, this bacterium can also cause intestinal illness and, occasionally, severe systemic infections and thus the products from cattle and sheep are suspected as sources of transmission  production as well as tolerance to glycine and NaCl, selenite reduction and resistance to antibiotics are the available biochemical tests currently employed as differential diagnosis of C. fetus  is restricted to the bovine reproductive tract, and is associated to the venereal disease bovine genital campylobacteriosis (BGC), whereas C. fetus fetus (Cff) is mainly intestinal and is usually related to sporadic abortion. To date, the bovine products are subjected to strict regulations by the World Organization for Animal Health (OIE) and must be tested for the presence of C. fetus subsp. venerealis before international trading (2S) production are two of the best biochemical performing tests. For example, Cff strains show 1% glycine resistance and produce H2S in L-cysteine (L-Cys) enriched media. By contrast, Cfv strains fail to grow in 1% glycine-containing media and to produce H2S  are frequently isolated in some countries such as USA, UK, South Africa and Argentina, which complicates their accurate identification . This L-Cys importer could be part of the Class 3 ABC-transporters  were obtained from bovine clinical samples at the Bacteriology Unit . Thirty of these clinical isolates were randomly-selected for this study. In addition, the strains Cfv 97/608, Cfv 98/25 and Cfvi 99/541 were also selected because of the availability of their whole genome sequences  with 1.25 IU/ml polymyxin B sulfate, 5 μg/ml trimethoprim, 10 μg/ml vancomycin and 50 μg/ml cycloheximide . The plates were incubated under microaerophilic conditions  for 72 h at 37 °C. C. hyointestinalis NCTC11562 and the field isolate C. sputorum 08/209 were grown under the same conditions. C. coli NCTC11353 and C. jejuni NCTC11392 were cultured on Blood-Columbia agar plates (Oxoid) under microaerophilic condition for 24 h at 42 °C.All the 2S production in 0.02% L-Cys enriched medium. We also tested 1.3%, 1.5% and 1.9% glycine tolerance. The isolates were identified as Cff if they reduced sodium selenite, produced H2S and showed sodium chloride tolerance and at least 1% glycine resistance.The classification of the subspecies was performed following standard protocols : sodium A rapid protocol (freeze-thaw cycles) was applied to obtain the DNA template as follows. A loopful of each culture was collected and resuspended in 250 µl of sterile deionized water. Two cycles of freeze and boiling (−80/95 °C) were performed and the cellular debris were discarded after a centrifugation step. Two µl of the supernatant was used as PCR-template. High quality genomic DNA was obtained using mini spin columns . DNA quality was tested using the Qubit 4 fluorometer  and further used for sequencing purpose.2), 0.25 mM of each dNTP, 0.1 μM of each primer, and 1.25 U Taq polymerase , nuclease-free water to reach a final volume of 25 µl and Campylobacter DNA template.One forward and two reverse primers  were designed for a multiplex-PCR format using open source Unipro UGENE 1.31  Fig. 1AFig. 1A. The touch-down amplification program consisted of an initial step at 94 °C for 3 min, 10 cycles at 94 °C for 1 min, followed by annealing temperatures starting at 55 °C for 1 min and decreasing 1 °C per cycle from 55 to 45 °C. Then, an extension step was performed at 72 °C for 1 min, followed by 30 cycles with an annealing at 51 °C, and a final termination step at 72 °C for 8 min.2S-producing strains), whereas a 310 bp product refers to Cfv strains (which contain a partly deleted operon and are non- H2S-producing strains). All the products were resolved in 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. The PCR-products were submitted to the UGB unit-INTA to confirm their identity through Sanger sequencing.Under these conditions, the absence of the expected product of 1,390 bp makes the interpretation of the PCR results easy. A product of 714-bp is indicative of Cff and Cfvi strains . The genomes were annotated using PROKKA  of the most productive agricultural areas of Argentina. Paired-end Nextera XT libraries were constructed and sequenced in a MiSeq sequencer . A quality trimming step was applied to raw reads using Trimmomatic . De novog PROKKA  and RASTg PROKKA . The assC. fetus strains  from 19 countries and different hosts  were screened to search for the target sequences of the primers designed for the L-Cys transporter-PCR protocol. These data included the three genomes obtained in this study  and 37 publicly available genomes from GenBank. Additionally, reads from C. fetus strains (n = 174) deposited in ENA database (https://www.ebi.ac.uk/ena/) were also assembled, as mentioned above, and subsequently analyzed as follows. The Primer map software (http://www.bioinformatics.org/sms2/primer_map.html) was used for global searching of Fwd, Rev1 and Rev2 primer sequences. Primer Map output is a textual map showing the annealing positions of PCR primers. Afterwards, several conditions were evaluated, including annealing of both primers of each pair and their orientation. The position of each target annealing site was employed to estimate the amplicon size. The program, by default, does not allow mismatches. Cases where the annealing was confirmed for a single primer were classified as not detected or unknown.In total, whole-genome sequence data of 214 2S production biochemical test and the L-Cys transporter-PCR was tested with Cohen´s Kappa statistic.The agreement between the HC. fetus isolates with distinct H2S-biochemical test results  strains was of 714 bp. This result coincided with a complete version of the L-Cys transporter operon and this pattern was named “CFF/CFVI.” Amplifications from Cfv strains generated a smaller product of 310 bp, equivalent to a partially deleted operon, and this profile was named “CFV”   . This reL-Cys transporter-PCR analysis displayed a perfect correlation with the H2S production test (κ = 1). The analysis of concordance between tests is shown in Table S2.The results from the C. fetus to further support this conclusion.We also addressed an in silico analysis of genomic sequences from mammalian L-Cys transporter-PCR in a more diverse panel of strains, we applied an in-silico PCR-strategy by performing searches of the primer targeting sequences in whole genomes of 214 C. fetus strains (three of which were obtained in this study by Next-Generation Sequencing technology). For this purpose, we employed the online Primer map application. The same products as the obtained by the wet lab-PCR were considered among all the predicted PCR products and the same patterns were determined according to the product size. This approach confirmed the primer annealing sites, and consequently, also allowed us to define the type of L-Cys transporter operon in 213 out of 214 C. fetus strains   . The ana2S-production test, 14 out of 43 strains that were identified as Cfv in the database did not match with the criteria of the deleted L-Cys transporter for this subspecies. Instead, these strains displayed a CFF/CFVI pattern  through the wet-lab and in silico-PCR approaches. The strains Cfv 97-608, Cfv 98-25 and Cfvi INTA 99/541 were selected from the lability . The L-Cdictions  and 2.L-Cys transporter-PCR and the H2S-production test for C. fetus analysis. Furthermore, it highlights the lack of consensus in the classification of these bacteria between the different laboratories around the world.Altogether, this study showed a strong concordance between the results of the To date, phenotypic tests are among the most valuable methods to identify and differentiate microorganisms. However, these tests are usually time-consuming, because they are growth-rate dependent, and the whole process depends on the objectivity and skills of the operator. Furthermore, a proper standardization, which is essential to obtain reliable and reproducible results, is often absent. Despite all this, the replacement of these phenotypic tests by molecular techniques is not always an alternative to date. The antimicrobial resistance constitutes a good example of complementary testing, and this particular phenotypic trait can be tested by bacteriological methods and at molecular level in several pathogens .C. fetus differentiation. For example, different studies have employed molecular techniques such as PCR based on different target genes to differentiate Cfv from Cff , the last subspecies proposed of C. fetus, has a complete version of the operon. This is the case of the strain Cft 03/427 (whose genome is the representative of the species) which has been concordantly described as an H2S-producing strain elsewhere , which has a long history in culturing this bacterium and in performing its biochemical based classification. In this way, the wet-lab approach showed a perfect correlation not only with the H2S production test, but also with the C. fetus subspecies. Indeed, a CFF-CFVI pattern, which is indicative of L-Cys complete transporter, was associated with H2S-producing strains typically classified as Cff or Cfvi. By contrast, a CFV pattern, which is indicative of a deleted transporter, was exclusively associated with H2S-non-producing strains, which are typically classified as Cfv.In this study, we tested 2S production test, but not with the reported subspecies of the strains.On the other hand, when we performed the in silico study, we analyzed genomic data from strains classified elsewhere by both molecular based approaches and/or biochemical tests and, as mentioned above, both techniques frequently displayed discordant results. In this way, we have obtained a perfect correlation with the H2S. This strain was included in this study and the PCR-L-Cys result was concordant with the phenotype of this strain. Later studies have also tested this strain and successfully sequenced its genome. Indeed, 2S positive . Later, 2S-producing strain. However, in this latter work, it has been called Cfvi 98-25 regardless the biochemical traits reported. In our study, the in silico sequence data analysis revealed the polymorphism of the L-Cys transporter (CFV pattern) typical of the non- H2S- producing strains.This discordancy is well reflected by the strain 98-25. Researchers from the Bacteriology Unit of INTA-Balcarce isolated this strain in 1998 from aborted foetus, and originally typed it as Cfv because of its glycine sensitivity and its inability to produce HTherefore, the same strain could display different biochemical traits when assayed in different labs -or time- and this is the classical bottle-neck of phenotypic tests.L-Cys transporter (CFF/CFVI pattern). Hence, at first glance, the hypothesis that all Cfv isolates do have a deleted L-Cys transporter appeared as not valid, according to the in silico analysis. However, when we searched the biochemical tests reported for some of these strains, we concluded that the in silico results presented here were concordant with the H2S production test. This discrepancy with the subspecies assigned could be due to the classification method of the strains that is frequently based on molecular techniques regardless the biochemical test results and moreover; the chosen method is not always specified . Although the presence of a T4SS has been previously associated to Cfv strains .Contingency table and calculation of the Cohen’s Kappa coefficient. Data set from Click here for additional data file.10.7717/peerj.7820/supp-3Supplemental Information 32S production test was included in the analysis. The analysis showed a perfect agreement (ĸ = 1) .Contingency table and calculation of the Cohen’s Kappa Coefficient. Data set from Click here for additional data file.10.7717/peerj.7820/supp-4Supplemental Information 4Click here for additional data file.


Helicobacter pylori (H. pylori), which is the main cause of gastric diseases such as chronic gastritis and stomach cancer. H. pylori adhesin A (HpaA), which is a surface-located lipoprotein, is essential for bacterial colonization in the gastric mucosa. HpaA had been proposed to be a promising vaccine candidate against H. pylori infection. However, the effect of non-lipidated recombinant HpaA (rHpaA) to stimulate immune response was not very ideal, and the protective effect against H. pylori infection was also limited. Here, we hypothesized that low immunogenicity of rHpaA may attribute to lacking the immunostimulatory properties endowed by the lipid moiety. In this study, two novel lipopeptides, LP1 and LP2, which mimic the terminal structure of the native HpaA (nHpaA), were synthesized and TLR2 activation activity was confirmed in vitro. To investigate whether two novel lipopeptides could improve the protective effect of rHpaA against the infection of H. pylori, groups of mice were immunized either intramuscularly or intranasally with rHpaA together with LP1 or LP2. Compared with rHpaA alone, the bacterial colonization of the mice immunized with rHpaA plus LP2 via intranasal route was significantly decreased and the expression levels of serum IgG2a, IFN-γ, and IL-17 cytokines in spleen lymphocyte culture supernatant increased obviously, indicating that the enhanced protection of LP2 may be associated with elevated specific Th1 and Th17 responses. In conclusion, LP2 has been shown to improve the protective effect of rHpaA against H. pylori infection, which may be closely related to its ability in activating TLR2 by mimicking the terminal structure of nHpaA.Over fifty percent of the people around the world is infected with  Helicobacter pylori (H. pylori) was identified as a category I human carcinogen by the International Agency for Research on Cancer in 1994. Infection with H. pylori was proved to be associated with peptic ulcer, chronic gastritis and gastric carcinoma. It is estimated that over half of the world's population is infected with H. pylori , also described as neuraminyllactose-binding haemagglutinin (NLBH), can bind to various glycosylation components on the surface of gastric epithelial cells, which is essential for colonization of the bacteria to gastric mucosa  could induce protection against  in mice –19. Howe limited , 21. Howpathways –25. In apathways –28. BaseH. pylori was evaluated and the mechanism was explored.It had been reported that lipoproteins could be recognized by TLR2 mainly depending on their N-terminal lipid moiety , 30. The2 was used for euthanasia.Animal maintenance and laboratory procedures were carried out according to the National Institutes of Health Guidelines for the Use of Experimental Animals and approved by the Medicine Animal Care Committee of the Third Military Medical University. Well-trained and skilled animal care personnel participated in the current study to minimize the suffering of animals. All procedures were performed under anesthesia with 1% sodium pentobarbital to alleviate pain. The health of the mice we monitored every 8–12 h and COSpecific-pathogen-free (SPF) female BALB/c mice aged 6–8 weeks old were purchased from the Experimental Animal Center of the Third Military Medical University, and were kept under pathogen-free conditions. HK-2 (human kidney 2) was purchased from the ATCC (American Type Culture Collection). HEK-Blue™-mTLR2 cells were purchased from InvivoGen  and cells were grown in DMEM supplemented with 10% FBS, L-glutamine (2 mM), Normocin (100 μg/mL), penicillin (50 U/mL), streptomycin (50 g/mL) and passaged when reached 70% confluence. Cells were scraped and then resuspended in HEK-Blue™ Detection medium . The induction of SEAP was detected at 620 nm by a spectrophotometer.E. coli and purified as previously described (Helicobacter Pylori Medium (HB8674) was purchased from Hopebio . HRP conjugated goat anti-mouse IgG (ab97200), IgG1 (ab97240), IgG2a (ab97245), and IgA (ab97235) were the products of Abcam . Mouse IFN-γ (DKW12-2000-096), IL-17A (DKW12-2170-096), IL-4 (DKW12-2040-096) precoated ELISA kit were purchased from Dakewe . Anti-mouse CD11c-FITC (117305), CD80-APC (104713), CD86-PerCP (105025) were the products of BioLegend .rHpaA was expressed in escribed  31, 32), 32E. co2Cys-OH was introduced to the N terminus of the peptide (SPHIIETNEV) linked to the resin. The Fmoc group was subsequently cleaved. The molecular were detached from the resin and all protecting groups were deprotected to yield the lipopeptide LP2.LP1 and LP2 were synthesized with solid-phase methods as previously described . LP1 wasHemolysis and cell viability assay was performed using methods described previously . BrieflyThe morphology of the synthetic lipopeptides was investigated by TEM negative staining technique. LP1 or LP2 at 100 μg/mL was deposited onto a copper grid and negative stained with 2% phosphotungstic acid for 30 s. After removal of excess of the solution and kept dried for at least 6 h, TEM images were taken by a JEOL JEM-1230  .4 cells) was added to the sample and then incubated at 37°C for 14 h. Samples were analyzed for SEAP activity after which OD was measured in a spectrophotometer at 620 nm (The sample was added to a flat-bottom 96-well plate (20 μL per well) and an equal amount of endotoxin-free water was used as a negative control. Followed by HEK-Blue™ mTLR2 cells were resuspended in the pre-warm HEK-Blue™ Detection, cell suspension   and IL-4 (20 ng/mL) at 37°C, 5% COFor the different delivery routes, mice were divided randomly into two sections. In the first section, mice were intranasally immunized 5 times in 4 weeks with PBS, rHpaA alone, or rHpaA plus one of the two synthetic lipopeptides (2 μg/mouse). The final amount of rHpaA for each intranasal immunization was 10 μg in 20 μL PBS. In the second section, the mice were immunized intramuscularly three times at intervals of 2 weeks with 50 μg of antigen in the same groups to the first section. On day 43, sera and intestinal lavage fluid were collected for further determination.9 BALB/c mouse-adapted H. pylori four times for 4 days. At 2-week post-challenge, the number of H. pylori colonization was quantified by real-time PCR , and then left untreated or treated with antiserum (1:100 dilution) at 4°C for 1 h. AGS cells were incubated with pretreated bacterial suspension at an MOI of 100 for each species. After 2 h of incubation, the cells were washed with PBS three times in order to remove unbound bacteria. The colonization of H. pylori attached to AGS cells was quantified by real-time PCR as described in section H. pylori Challenge and Quantification.The escribed , 42. Brip-value ≤ 0.05 were considered statistically significant., Bonferroni test were further performed if inter-group differences were statistically significant, with the significant level α' = α/k (k is the number of comparison times).All statistical analysis were conducted by the SPSS 21.0 statistical package. Data were expressed as means ± standard deviation (SD). One-way ANOVA was used to analyze inter-group differences, and differences with a http://www.mrc-lmb.cam.ac.uk/genomes/dolop) and UniProt (https://www.uniprot.org) database, nHpaA has two lipidative forms, monopalmitoylation (Pam) and dipalmitoylation (Pam2). The peptide containing 10 amino acids (CSPHIIETNEV), derived from the N terminus of the nHpaA, was selected for chemical coupling with Pam and Pam2, respectively, resulting in two novel lipopeptides  expression were significantly elevated in LP1 and LP2 stimulation group compared with PBS group . LP2 shoin vitro, we further investigated whether vaccinated with rHpaA and the synthetic lipopeptides could provide more potent protection against H. pylori. BALB/c mice were intramuscularly or intranasally immunized with rHpaA plus LP1 or LP2, and then orally challenged with H. pylori strain B0. Bacteria colonization in mice stomachs were determined by real-time PCR  and multi-component vaccines   , and proH. pylori infection? Recent studies suggested that high levels of IgG antibodies may not be necessary for vaccine protection  is rich in APCs, especially mucosal DCs which express TLR on the surfaces (Though both LP1 and LP2 were synthetic lipopeptides based on the structure of nHpaA, there was significant difference in assisting rHpaA to provide protection against immunity . Previousurfaces , 57. SynH. pylori infection via intranasal immunization, which might be mediated by specific Th1 and Th17 responses. In future studies, the mechanisms which were involved in the immune-stimulating activity of LP2 will be further investigated. Other antigens will be administered in combination with LP2 to investigate whether LP2 also could enhance their ability to confer protection against H. pylori. For the improvement of antigen specific immune response, co-delivery system that delivers both the antigen and LP2 will be designed in the following studies.In this study, the synthetic lipopeptide LP2 was proved to enhance the protective effect of rHpaA against https://www.mrc-lmb.cam.ac.uk/genomes/dolop.Publicly available datasets were analyzed in this study. This data can be found here: This study was carried out in accordance with the recommendations of the Animal Ethical and Experimental Committee of the Third Military Medical University. The protocol was approved by the Animal Ethical and Experimental Committee of the Third Military Medical University.H-BL and Q-MZ designed experiments. R-YX, M-FG, LG, CL, SL, LJ, LN, C-JM, and D-QC carried out experiments. H-BL, JL, ZJ, and SS analyzed experimental results. H-BL, Q-MZ, R-YX, and MG wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cordyceps militaris (C. militaris) were evaluated in this study. The results of gas chromatography (GC) and high-performance gel permeation chromatography (HPGPC) showed that a higher extraction temperature could degrade the polysaccharides with 188 kDa, mainly composed of glucose, and increase the dissolution rate of polysaccharides about 308 kDa, mainly consisting of rhamnose and galactose. In addition, the CMPs displayed the same sugar ring and category of glycosidic linkage based on Fourier-transform infrared spectroscopy (FTIR) analysis, however, their invisible structural difference occurred in the specific rotation and conformational characteristics according to the results of specific optical rotation measurement and Congo red test. In vitro antitumor experiments indicated that CMPs-4 possessed stronger inhibitory effects on human esophagus cancer Eca-109 cells by inducing cell apoptosis more than CMPs-80 did. These findings demonstrated that the polysaccharides extracted with cold water (4 °C) could be applied as a novel alternative chemotherapeutic agent or dietary supplement with its underlying antitumor property.The effects of different extraction temperatures (4 and 80 °C) on the physicochemical properties and antitumor activity of water soluble polysaccharides (CMPs-4 and CMPs-80) from  C. militaris, an entomogenous fungus belonging to the class Ascomycetes, has been widely used as a folk tonic food and therapeutic drug for various diseases in East Asia [C. militaris, known as the Chinese rare caterpillar fungus, has a similar pharmacological effect to the well-known Chinese traditional fungus Cordyceps sinensis [C. militaris [C. militaris possess multiple biological properties, such as antioxidant [ast Asia . C. milisinensis . Althougilitaris ,4. Moderioxidant ,6,7, antioxidant ,9, immunioxidant ,11,12, aioxidant , anti-inioxidant , etc.Cordyceps gunnii mycelia polysaccharides, and their remarkable differences were observed in their antitumor effects and physicochemical properties, such as scanning electron microscopy, intrinsic viscosity, and specific rotation [In general, the physicochemical properties of polysaccharides using conventional water extraction were strongly affected by multiple parameters, such as the source of raw materials, water temperature, extraction time, extraction solvent, ratio of liquid to solid, and so forth ,16. In arotation . Zhang erotation . Zhao etrotation . TherefoC. militaris polysaccharide have mainly focused on the optimization of extraction conditions for maximum yield and good bioactivity, however, the influence of extraction temperature on the physicochemical properties and antitumor activities of polysaccharides remains limited. In this study, the main objective was to investigate the effects of cold- and hot-water extraction from C. militaris on the relationship between the structural characterization and antitumor activities of polysaccharides, and to further evaluate their antitumor capacities by comparing the in vitro inhibition effects on human esophagus cancer Eca-109 cells. The obtained CMPs were named CMPs-4 and CMPs-80, respectively, according to their extraction temperature (4 and 80 °C). Recently, researches concerning the The sugar and protein contents, and their molar ratio of polysaccharide samples, are shown in y = −0.3866x + 8.8472, R2 = 0.9904 . The retention time, molecular weight population, and relative contents of CMPs-4 and CMPs-80 are displayed in The regression equation of the standard dextran curve was established as follows: −1 attributed to O–H stretching vibrations, a weak band at around 2932 cm−1 due to C–H stretching vibrations, and a band at around 1404 cm−1 attributed to the C–H bending vibrations. In addition, the strong absorption band at around 1625 cm−1 was derived from C=O stretching vibrations. The absorption bands in the range of 1144–1048 cm−1 could be assigned to the stretching and bending vibrations of C–O, C–C and C–O–C, and C–O–H, indicating the presence of pyranose rings, and the band at around 845 cm−1 suggested the presence of α-type glycosidic linkages in CMPs. This observation was consistent with the results of Zhu et al. [C. militaris, mainly contained mannose, glucose, and galactose with α-type glycosidic linkages. The IR spectrums of CMPs-4 and CMPs-80 were almost identical only with a slight difference in the intensity of bands, indicating that different extraction temperatures had no distinct effect on sugar rings or the category of glycosidic linkages of polysaccharides, which coincided with the analysis of monosaccharide composition.IR spectra of CMPs extracted with different temperatures showed very similar absorption bands . Three cu et al. , who indThe specific optical rotations of CMPs-4 and CMPs-80 were +49° and +52°, respectively, which showed the presence of α-glycosidic bonds in these two polysaccharides. The difference in specific optical rotations for both CMPs-4 and CMPs-80 also indicated that different extraction temperatures could cause changes in the stereochemical structure of polysaccharides, which might influence the strength of bioactivities. max) of Congo red in the absence or presence of CMPs-4 and CMPs-80. The results showed that the λmax of Congo red CMP complexes exhibited a significant transition into longer wavelengths in the weak alkaline solution (<0.20 M NaOH), indicating the existence of a triple helix configuration in both CMPs-4 and CMPs-80. However, when the NaOH concentration was higher than 0.2 M, the λmax of Congo red CMP-80 complexes declined sharply, which might be due to the shift of space spiral structures to single flexible chains [max with the alkaline concentration increasing, which is probably because the CMP-4 fractions extracted with cold water could maintain a stable triple helical structure in the high concentration of alkaline solutions. Further research is still needed to explain the phenomenon.Congo red, an acid dye, can interact with polysaccharides with triple-helical conformations to form complexes, leading to a bathochromic shift of the absorption maximum . Figure e chains . For CMP50 value of CMPs-4 at 24 h was calculated to be 532.9 μg/mL. In contrast, CMPs-80 showed a weaker inhibition (about <30% inhibitory rate) against human hepatoma Eca-109 cells than CMPs-4. The above data suggested that the inhibition ability of these polysaccharides was dose-dependent, and higher temperatures would reduce the antitumor activity of polysaccharides due to the damage to their structures.An MTT assay was applied to compare the inhibitory effect of CMPs-4 and CMPs-80 towards human esophagus cancer Eca-109 cells. As shown in To examine whether the tumor cells’ growth suppression was caused by the induction of cell apoptosis, Eca-109 cells were treated with CMPs-4 for 24 h and the morphological changes were observed under an inverted light microscope A. The reThe CMP-4-induced cell apoptosis was further evaluated and quantified using flow cytometry analysis after Annexin V-FITC/PI staining. Seen from Nuclear morphology analysis can further confirm cell apoptosis by Hoechst 33,258 staining. As shown in Esophageal cancer (EC), a highly malignant carcinoma, is estimated to be the eighth most common cancer and the sixth leading cause of cancer death worldwide . CurrentAstragalus membranaceus exhibit the stronger antitumor and immunoregulation activities, with minimal side effects due to the degradation of active constituents by higher temperature [C. militaris with two extraction temperatures (4 and 80 °C) were deeply researched. Results showed that monosaccharides’ molar ratio and the molecular weight distribution of these two polysaccharides exhibited significant discrepancies, where CMPs-80 had higher proportions of rhamnose and galactose, but a lower proportion of glucose. In addition, the CMPs-80 showed a higher molecular weight (about 308 kDa) with a relative high percentage area (62.46%), while CMPs-4 had a component about 188 kDa with a percentage area (53.23%). These results indicated that higher extraction temperature could degrade the polysaccharide mainly composed of glucose and increase the dissolution rate of polysaccharides consisting of rhamnose and galactose. The IR spectrums, specific optical rotation analysis, and conformational characteristics showed no significant difference between these two polysaccharides.Temperature, as a critical parameter, influences various biological activities in animals and plants . Currentperature . In the C. militaris is responsible for the antitumor activity, which need to be further researched in the future.Apoptosis is an important method for multicellular lives to maintain homeostasis and a key survival process for hosts when it occurs only tumor cells ,33. CharC. militaris were obtained from the incubation base of industry-university-research cooperation in YiLi Normal University , and were first washed with deionized water thoroughly to remove all dirt and other contaminants. The C. militaris were dried to constant weight in an oven at 50 °C, screened through an 80 mesh sieve to obtain the homogeneous powder sample, and finally stored under suitable conditions for subsequent study. Fetal calf serum (FCS) was from Hangzhou Sijiqing Co. . Annexin V-FITC/PI Apoptosis Detection Kits were purchased from Beyotime Biotechnology Co. . 5-fluoro-2,4pyrimidinedione (5-Fu), 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich . Other chemicals and agents were of analytical grade.C. militaris powder (50 g) was extracted with distilled water  at either 4 or 80 °C for 4 h. After extraction, the water extracts were filtered and centrifuged  to remove the insoluble fractions. The collected supernatant was concentrated by freeze-thaw method and then precipitated by the addition of 4 volumes of absolute ethanol overnight at 4 °C. The precipitated polysaccharides were obtained after centrifugation at 4500 rpm for 10 min, and washed using anhydrous ethanol and acetone alternately 3 times to remove the lipids and pigments thoroughly. Afterwards, the crude samples were re-dissolved in deionized water, and the protein was removed according to the Sevag method with 1-butanol and chloroform  for 5 times [Mw 1000) in distilled water for three days to exclude the salt and low molecular weight sugars, and lyophilized to get the C. militaris polysaccharides (CMPs) for further study.The extraction procedure was carried out using water extraction and alcohol precipitation. The dried  5 times . Finallyd-glucose as a standard [The contents of carbohydrate in the CMPs-4 and CMPs-80 were determined according to the phenol-sulfuric acid method using standard . The prostandard .l-rhamnose, d-Arabinose, d-Xylose, d-Mannose, d-Glucose, and d-Galactose were also derivatized as the standards to identify the monosaccharide components of the samples by comparing the retention times.The monosaccharide composition of the polysaccharide samples was determined after acid hydrolysis and derivatization using GC  according to the procedures described previously . In brieThe molecular weight distribution of CMPs-4 and CMPs-80 was performed by HPGPC  equipped with a TSK-gel G4000 PWxl column  and Refractive Index Detector  based on the approaches described previously . Twenty −1 on a FT-IR spectrophotometer .The characteristic groups of the polysaccharide samples were identified using FT-IR. A total of 0.7 mg of dried polysaccharide samples were blended with 150 mg of dry KBr powder, followed by pressing into a KBr disk for further research. The IR spectra was collected in the range of 4000–400 cmt is the detecting temperature, λ is the wavelength of light source (λ = 589 nm), L is the length of polariscope tube, and C is the polysaccharide solution concentration (g/mL).The digital automatic polarimeter  was applied to determine the optical rotation of the polysaccharide samples. The 10 mg CMPs-4 and CMPs-80 were dissolved with 10 mL distilled water, respectively, and transferred into the polariscope tubes. The optical rotation of the samples was read directly with an automatic polarimeter at the designated temperature (20 ± 0.1 °C). The specific optical rotation was calculated according to the following equation: The conformation of the CMPs-4 and CMPs-80 was evaluated following the method reported by Su et al.  with lit2, and 100 μL of cell suspension (1× 105 cells/mL) was gently seeded into 96-well cell culture plates and incubated overnight. Subsequently, Eca-109 cells were subjected to different concentrations of sterile polysaccharide solutions  and incubated at 37 °C in a humidified 5% CO2 incubator for 24 h, followed by the addition of 20 µL of MTT (5 mg/mL in PBS) into each well. After 4 h of incubation, the supernatant was removed by centrifugation at 1000 rpm for 10 min and the precipitated formazan was dissolved in 150 µL/well of DMSO. 5-Fu was used as the positive control. The absorbance value was detected at 570 nm using a microplate reader . The inhibition effect of the polysaccharides on Eca-109 cells was calculated as follows: Inhibitory ratio (%) = (1 − ATreated/AControl) × 100.The viability of Eca-109 cells was evaluated in vitro using an MTT assay as previously described , with mi4 cells/mL) were inoculated into 6-well plates and treated with different concentrations  of CMPs-4. After 24 h, the morphological changes of Eca-109 cells were observed under an inverted optical microscope .Eca-109 cells (8 × 104 cells/mL) cultured in 6-well plates were treated with an Annexin V/PI Apoptosis Detection Kit according to the directions. After treatment with the designated concentrations  of CMPs-4 for 24 h, the cells were collected and stained with the Annexin V-FITC/PI apoptosis detection kit following the manufacturer’s manual. In brief, the harvested cells were incubated with 100 μL of a 1 × binding buffer containing 5 μL Annexin V-FITC and 5 μL propidium iodide (PI) for 10 min at room temperature in the dark. The stained cells were detected immediately by flow cytometry .Eca-109 cells (8 × 104 cells/mL) were seeded into the 6-well cell culture plates and exposed to various concentrations of CMPs-4  for 24 h. Subsequently, the cells were collected, washed with cold PBS, and stained with Hoechst 33258 solution at room temperature for 15 min. After that, the stained cells washed in PBS and observed by inverted fluorescence microscope .Eca-109 cells .All values were expressed as the mean ± standard deviation (S.D.) and the statistical significance of differences was determined using the student’s t-test and one-way analysis of variance (ANOVA). The data was considered significant at C. militaris with water under two different temperatures , and researched the relationship between the structural characteristics and antitumor effects. Our results showed that higher extraction temperatures could degrade the polysaccharide with 188 kDa, mainly composed of glucose, and increase the dissolution of polysaccharide with 308 kDa, mainly consisting of rhamnose and galactose. The in vitro antitumor test showed that CMPs-4 possessed stronger antitumor activity than CMPs-80, which could significantly inhibit the proliferation of Eca-109 cells via inducing their apoptosis. This study provides a novel extract method to obtain polysaccharides with higher bioactivity and a theoretical basis for the application of CMPs-4 in food and medical industries.In conclusion, we extracted the polysaccharides from